2025 Theses Doctoral

Rewiring of cell signaling by pathogenic mutations in the tyrosine phosphatase SHP2

Van Vlimmeren, Anne Elise

The protein tyrosine phosphatase PTPN11/SHP2 is an important hub in many crucial cell-signaling pathways. SHP2 consists of a catalytic protein tyrosine phosphatase (PTP) domain and two phosphotyrosine-recognition domains (N-SH2 and C-SH2), which regulate SHP2 localization and activation. SHP2 exists primarily in an auto-inhibited state where the N-SH2 domain blocks access to the catalytic site on the PTP domain. SHP2 can be activated by phospholigand engagement of the SH2 domains, which induces a conformational change that rotates the N-SH2 domain away from the catalytic pocket.

Mutations in SHP2 are associated with several types of cancer, particularly leukemias, and multiple congenital disorders. Many mutations in SHP2 disrupt the auto-inhibitory interactions between the N-SH2 domain and PTP domain, which causes an increase in SHP2 basal catalytic activity and enhances oncogenic signaling in cells. However, several disease-causing SHP2 mutations do not conform to this model, suggesting additional, alternative mechanisms through which SHP2 mutations drive pathogenic signaling. The broad goal of this work is to elucidate different molecular mechanisms of pathogenicity across disease-relevant SHP2 variants through biochemical, biophysical, cell biological, proteomic, and transcriptomic approaches.

Moreover, we use these insights to gain deeper understanding of the functional role of SHP2 in cell signaling.We first characterize five mutations near the SH2 binding pockets and show that the T42A mutation rewires the interaction specificity of the N-SH2 domain. Our biochemical validation experiments and molecular dynamics analyses reveal that this mutation induces a distinct mode of phospholigand binding and implicate a nearby lysine residue in the T42A-driven change in ligand binding preferences. We additionally show that the T42A mutation enhances SHP2-dependent signaling in cells. Proteomic analyses using TurboID-proximity labeling across multiple SHP2 variants demonstrate that different mutations remodel the SHP2 interactome in unique and context-dependent ways. We also show that disease-mutations in SHP2 can alter subcellular localization, with several mutants increasingly localizing to the mitochondria.

To understand how these shifts in protein-protein interactions are paralleled by transcriptional consequences, we profiled the transcriptomes of 15 pathogenic SHP2 variants using single-cell RNA-sequencing. Our analysis revealed that a perturbation to the C-SH2 domain of SHP2 approximates the transcriptional profile of SHP2 knock-out cells, highlighting the importance of the scaffolding and regulatory functions of SHP2. We also identified convergence between distinct mutations on similar transcriptional programs and demonstrate that this is likely caused by shared altered substrate-binding preferences.

Emerging from these analyses is a model of SHP2 as a multifunctional signaling integrator, in which disease-associated mutations disrupt not only regulation of catalytic activity, but also spatial distribution, binding specificity, and scaffolding capacity. This idea is further underscored by our work characterizing a not previously reported active conformation of SHP2. This novel state, stabilized by the E139D mutation, is constrained in its range of potential SH2 interactors, providing another mechanism through which SHP2 mutations rewire signaling. Together, this work reveals diverse and mutation-specific mechanisms through which SHP2 dysregulation drives aberrant signaling. By integrating multi-scale analyses, it provides foundational insights into the molecular mechanisms of SHP2 function and pathogenicity, with implications for both basic signaling biology and therapeutic targeting.